The drug efux pump is a crucial mechanism implicated in resistance to multiple antimicrobials. Thymoquinone (TQ) has evidently demonstrated multiple activities, antibacterial being the most efective. Knowledge about TQ activity against multidrug-resistant Staphylococcus aureus is very scarce. Therefore, the present study was conducted to investigate TQ resistance modulation in ciprofoxacin (CIP) and doxycycline (DO) multidrug-resistant S. aureus. Forty-seven samples were collected from diferent sources, and S. aureus was isolated and identifed. Then, S. aureus resistance profles to antimicrobials, N. sativa essential oil, and TQ; the correlation between TQ-MIC readings and disc difusion; cartwheel and ethidium bromide (EtBr) accumulation assays; and norA gene expression were all described within silico molecular docking for TQ interactions with norA efux pump protein. TQ-MICs ranged from 5–320 µg/ml. TQ down-regulated norA gene expression, resulting in a drop in efux pump activity of 77.5–90.6% in the examined strains, comparable to that observed with verapamil. Exposure of S. aureus strains to CIP and DO raises the initial basal efux pumping expression to 34.2 and 22.9 times, respectively. This induced efux pumping overexpression was substantially reduced by 97.7% when TQ was combined with CIP or DO. There was a signifcant reduction of MICs of CIP and DO MICs by 2–15 and 2–4 folds, respectively, after treatment with 0.5XMIC-TQ in resistance modulation assays. These results refer to TQ ligand inhibitory interactions with NorA protein in molecular docking. Interpretations of inhibition zone diameters (IZDs) of disc difusion and TQ-MICs exhibit independence of MICs from IZDs, as indicated by invalid linear regression analysis. TQ signifcantly reduced efux pumping S. aureus induced by CIP and DO, but further investigations are needed to improve TQ-pharmacokinetics to restore CIP and DO activity and suppress fuoroquinolone and doxycycline-resistant S. aureus selection in clinical and animal settings.